Enhygrofungin and process for preparing the same

ABSTRACT

Antibiotic enhygrofungin producible by culturing Streptomyces hygroscopicus var. enhygrus var. nova in an aqueous nutrient medium. Enhygrofungin inhibits the growth of various fungi. For example, it is active against Cryptococcus neoformans and can be used to treat pigeon roosts to inhibit this fungus which has been found in pigeon droppings.

United States Patent Bergy et al. 1 Oct. 17, 1972 [54] ENHYGROFUNGIN AND PROCESS FOR [56] References Cited A PREPARING THE SAME [72] I t M I I E B H H k UNITED STATES PATENTS nven ors: see In er erman oe L R 2,746,902 5/1956 Gottlieb et al ..424/1 15 e 3 118 812 l/l964 Gaeumann etal 424/121 Kalamazoo, Mich. g

Primary Examiner-Albert T. Meyers [73] Asmgnee: h Up-Mm Comimny, Kalamazoo Assistant Examiner-Daren M. Stephens Mlch- Attorney-Roman Saliwanchik and John Kekich [22] Filed: June 13, 1969 [57] ABSTRACT [2 1 App] No 833l35 m, Antibiotic enhygrofungin producible by culturing Streptomyces hygroscopicus var. enhygrus var. nova in an aqueous nutrient medium. E'nhygrofungin inhibits the growth of various fungi. For example, it is active against cry tococcu neoformans and can be used to [51] Int. Cl. ..A61k 21/00 treat pigeon roosts to inhibit this fungus which has [58] Field of Search ..424/ l 21 been found in pigeon droppings.

7 Claims, 3 Drawing Figures PAIENTEDncm 1912 v 3.699.223

sum 2 or 3 7 III MALCOLM E. BERGY HERMANHOEKSEMA LEROY E. JOHNSON NTORS ATTORNEY PATENTEDHBI 1 i912 3,699,223

' SHEE-I 3 BF 3 MALCOLM E. BERGY HERMAN HOEKSEMA LEROY E. JOHNSON INVENTORS ATTORNEY ENIIYGROFUNGIN AND PROCESS EOR PREPARING THE SAME BRIEF SUMMARY OF THE INVENTION Enhygrofungin (U29,479) is an amphoteric nonpolyenic chemical compoundwhich is producible by culturing an enhygrofungin-producing actinomycete in an aqueous nutrient medium. It has the property of adversely affecting the growth'of Grampositive bacteria,

tibiotic agents to prevent the growth of or reduce the number of bacteria and fungi, as disclosed above, in various environments. For example, it can be used as the anti-fungal agent in the shoe uppers disclosed in U.S. Pat. No. 3,130,505.

Enhygrofungin is considered to be a component of the known polyenic antibiotic complex endomycin. A process for the production of the endomycin complex, as well as its characterization is disclosed in U.S. Pat. No. 2,746,902. Enhygrofungin, a non-polyenic compound, is not disclosed in U.S. Pat. No. 2,746,902 as being a component of the endomycin complex. Accordingly, there is no disclosure of the properties of enhygrofungin nor of the manner for isolating enhygrofungin from the endomycin complex. Though the endomycin complex is known in the antibiotic art as having good antifungal activity, it has not been used medically because a standardized preparation could not be made. Preparations of endomycin complex would vary'depending on the amount of any particular component of the complex which was present. The inability to obtain a standardized preparation of the endomycin complex disclosed in U.S. Pat. No. 2,746,902, which could be usable for medical purposes, led to the work resulting in the discovery of the subject compound. In contrast to the non-crystalline polyenic endomycin complex, enhygrofungin is a crystalline nonpolyenic entity which is highly reproducible, and, therefore, can be standardized for medical uses. The fermentation conditions, including the microorganism, described herein, are a distinct improvement over those described in U.S. Pat. No. 2,746,902 to produce larger amounts of enhygrofungin in the endomycin complex.

DETAILED DESCRIPTION OF THE INVENTION CHARACTERIZATION OF ENHYGROFUNGIN Crystalline enhygrofungin has the following chemical and physical properties:

Color: white Elemental Analysis:

Found: C, 60.95; H, 9.00; N, 4.01; O, 16.46

Titration data: In glacial acetic acid with perchloric acid, equivalent weight 1,268 (does not titrate in T- butanol with pyridine).

Optical Rotation: [11:1 (c, 0.5 percent in dimethyl-formamide).

Solubilities: Enhygrofun gin is soluble at a level of less than 5 mg./ml. in water, acetone, ethyl acetate, methyl ethyl ketone, methylene chloride, chloroform, butanol, and ether. It is soluble at a concentration greater than 10 mg./ml. in methanol, ethanol, n-propanol, glacial acetic acid, acetone-water (3:2), and water saturated l-butanol.

Melting point: 119.3 C.

U.V. Absorption Spectra:

Methanol:

lnflection at 227 mu, a 25.77

- max. at 231 m;r,a=27.22

Slight inflection at 240 mp, a 17.74 0.1 N I-ICI in methanol: lnflection at 226 mp, a 25.96

4 max. at 231mu,a=27.34

Slight inflection at 240 mg, a 17.97

0.1 N KOH in methanol; lnflection at 227 mu, a 25.70 I max. at 231 m;r,a=27.17

Slight inflection at 240 my, a 18.02

Infrared Spectrum: The infrared absorption spectrum of enhygrofungin suspended in mineral oil mull is reproduced in FIG. 1 of the drawing. Enhygrofungin shows peaks at the following wavelength expressed in reciprocal centimeters:

The infrared absorption spectrum of enhygrogungin v in KBr from chloroform shows peaks at the following wavelengths expressed in reciprocal centimeters:

3370 (S) 1640(5) 1135 (S) .3200 (S) 1595 (S) 1085 (S) 2980 (S) 1455 (S) 1060 (S) 2930 (S) 1415 (S) 1025 (Sh) (S) 2890 (S) 1380 (S) 985 (S) 1715 (S) 1275 (broad) (S) 915 (M) 1660 (S) 1180 (M) 845 (M) Infrared absorption band intensities throughout this disclosure are indicated as S," M, and -W, respectively, and are approximated in terms of the backgrounds in the vicinity of the bands. An S band is of the same order of intensity as the strongest in the spectrum; M bands are betweenv one-third and twothirds as intense as the strongest band, and W bands are less than one-third as intense as the strongest band. These estimates are made on the basis of a percent transmission scale. Also, the designation sh appearing after a band reading denotes a shoulder type of band.

Papergram: The paper chromatographic pattern of enhygrofungin in the following solvent systems is as shown in FIG. 2 of the drawing:

1. l-butanol, water (84:16), 16 hours.

2. l-butanol, water (84:16) -l- 0.25 percent ptoluene-sulfonic acid, 16 hours.

3. l-butanol, acetic acid, water (2:1 :1 16 hours,

4. 2 percent piperidine (v/v) in l-butanol, water,

(84:16), 16 hours. 5. l-butanol, water (4:96), 5 hours. 6. l-butanol, water (4:96), 0.25 percent p-toluenesulfonic acid, 64 hours. The microorganism S. cerevisiae used in the papergram analysis was grown on agar consisting of the following nutrients:

Nuclear Magnetic'Resonance (NMR): (also known as Proton Magnetic Resonance) Enhygrofungin has a characteristic NMR spectrum as shown in FIG. 3 of the accompanying drawing. The NMR. Spectrum was observed on a Varian A- 60 sprectrometer on a solution (ca. 0.5 ml., ca. 15 percent concentration) of the sample of enhygrofungin in deuterated dimethylformamide.

. The spectrum was calibrated against internal tetramethylsilane and the precision of the A v was :t 1 c.p.s. Frequencies were recorded in c.p.s. downfield from tetramethylsilane.

The Antibacterial Properties of Enhygrofungin:

Enhygrofungin has antibacterial activity as shown in the following table. The test is a two-fold broth dilution test in brain heart infusion broth wherein a final dilution of a fully grown culture in brain heart infusion of 1/40,000 is made. Those organisms requiring blood are cultured in this but the final antibiotic test is made in the absence of blood, since it has been found that adequate growth is obtained without it. Incubation is unshaken at 37 C. End points read at 20 hours.

Brain heart infusion broth (Difco) has the following composition:-

Calf brains, infusion from Beef heart, infusion from 250 g. Proteose Peptone, Difco g. Bacto-dextrose 2 g. Sodium chloride 5 g. Disodium Phosphate 2.5 g. Water 1000 ml.

Minimum Inhibitory Test Organism Concentration (meg/ml.)

Staphylococcus aureus 8.0 Streptococcus hemolyticur 31.0 Streptococcus faecalis 31.0 Bacillus sublilis 4.0 Diplococcus pneumoniae 16.0

Requires blood for growth following table. The antifungal spectrum was determined by an agar dilution plate assay.*

Concentration of Enhygrofungin Test Organism in meg/ml.

Nocardia asteroides Blasmmyces dermatitidis C occidiodes int m in: Geotrichum .rp. Hormodendrum compactum Phr'alaphora verrucosa C ryptococcus neoformans Hisraplasma capsularum Sporoirichum schemrkii Monorporium apiospcrmum Trichophyton rubrum Trichophylon inrerdigitalc Candida albicans ABBOTT Trichophyton violaceum Trichophyron mentagrophyres Microsporum cam: Trichophyton asteroides Note:

inhibition 1 Partial Inhibition f No Inhibition Agar used consists of the following:

Difco Bacto-dextrose 10 g. Difco Bacto-peptone 5 g. Difco yeast extract I l g. Triple-distilled 11,0 to 1 liter pH 6.8 A'gar 15 g.

THE MICROORGANISM The actinomyceteused according to this invention for the production of enhygrofungin is Streptomyces hygroscopicus var. enhygrus. One of its strain characteristics is the production of enhygrofungin. A subculture' of the living organism was deposited without restriction and can be obtained from the permanent collection of the Northern Utilization and Research Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, U.S.A. Its accession number in this repository is NRRL 3664.

The microorganism of this invention was studied and characterized by Alma Dietz of The Upjohn Research Laboratories.

Streptomyces hygrascopicus var. enhygrus is a new soil isolate of the genus Streptomyces which has been found to differ in certain characteristics from the type culture Streptomyces hygroscopicus CBS (Centraalbureau voor Schimmel) notably in its production of the antibiotic enhygrofungin, and from the type culture Streptomyces endus NRRL 2339 (in its cultural characteristics). Streptomyces endur has been shown to have the main characteristics of the hygroscopicus type [Tresner, H. D., and E. J. Backus, Applied Microbiol. 4:243-250 (1956)], [Dietz, A., and J. Mathews, Applied Microbiol. 10:258-263 (1962)]. Minor distinguishing characteristics of the new variety may be noted by referring to the Tables following.

Streptomyces hygroscopicus var. enhygrus is compared with the type species Streptomyces hygroscopic-us, (Jensen) Waksman, CBS, and the type species Streptomyces endus NRRL 2339.

Color characteristics.

Aerial growth white to gray-white or gray-cream to gray. Moist, black, hygroscopic patches on some media. Melanin-negative. Appearance on Ektachrome is given in Table 1. Reference color characteristics are given in Table 2. The cultures may be placed in the White (W) and Gray (GY) color series of Tresner and Backus [Applied Microbiol. 11:335-338 (1962)]. Microscopic characteristics. I

Sporopl'lores in tight spirals. Sporophores spiralin the sense of .Pridham et al. [Applied Microbiol. 6:52-79 (1958)]. Spores frequently forming dark,

Table 5 TABLE l hygroscopic patches. Spores smooth with an irregular posslbly warty-surface by direct electron microscope Appearance of S. hygroscopicus cultures on examination. Spore surface moral-like when examined Ektachrome* by the carbon replication method of Dietz and s s s Mathews [Applled Mlcroblol. 10:258-263 (l962); Aphygros'mpicus hygroscopic plled MlClOblOl. 161935-941 (1968)]- Agar Medium val. enhygrru CBS NRRL Cultural characteristics. 2339 See Table 3. Carbon utilization. Bennett's I: (Y'irrltly YGi'fiY-Whlte gray e OW-lflll 6 CW 311 The abllrty of the culture to grow on carbon com- Capers mm: 5 I Gray pounds was determined in the synthet c medium of whlle Pridham and Gottlieb J. Bacteriol. '56: 107-114 R y only Yellowe r s y )]l ng. E- B Maltose-tryptone S Trace Trace Trace and D. Gottlieb, International Journal of Systemic Bacare; terlology 16:313-340 (1966)]. See Tables4 and 5. 1 R Yellow Yellow Yellow Temperature- Peptone-iron S Trace All cultures grow moderately well to well at tempera- 1K3 tures of l8-27 C. on Bennetts, Czapeks Sucrose, and 1 a Yellow Yellow Yellow 0.1% t rosine S Trace Trace Trace Maltose tryptone agars Trace to fan vegetative growth Y Braywhite graymhite gray occurs m 24 hours at 55 C. white The characteristics of Streptomyces hygroscopicus 25 R Red Red Pale var. enhygrus var. nova, NRRL 3664, are given in the PM mam following tables: Casein starch S Gray Gray White R Gray Gray Pale Yellow Table 1 Appearance of S. hygroscopicus cultures on Ektachrome Table 2 Reference Color Characteristics of S.

hygmscopicus culturesv Dietz, A., "Ektachrorne Transparencies As Aids in Actlrlomycete- Table 3 Cultural Characteristics of S. hygroscopicus Classification, Annals of the New York Acad. of Sciences,

cultures 152-154, 1954. Table 4 Utilization of Carbon Compounds in the S Surface Synthetic Medium of Pridham and Gottlieb RE Reve.rse

TABLE 2 [Reference color characteristics of S. hugroacoptcus cultures] Color harmony manual, 3rd ed. 1948 1 NBS circular 553, 1955 2 S. hwro- S. S. acopicua hygroendus S. S. var. scopicua NRRL hwroscopicus hugroacoprcua S. endus Agar medium enhugrus CBS 2339 var. emwgrus CBS NRRL 2339 S b (m) 1 to (g) 21g (111) 263 m, 264 g 112 m, 122 g 110 g, 112 m Bennett's R 2 go (111) 2 go (g) 2 ge (g) gm 90 gm 94 m, 109 gm P 2 cc (g) 21a (g) 90 gm 91 gm, 94 g,

S 21a (g) 3 to (m) 0 (g) 94 g 112gm 63 gm 264 gm Czapek's sucrose "{1 2 dc (g) 1 to (m) c (g) 93 in 112 m, 122 g 261 gm 8 b (m) 2 eh (m) 1 do (g) 263 m, 264 g 92 m, 93 gm 121 m Maltose-tryptone g. 1 ca (5;) 2 cc (g) 2 ob (g) 104 g, 121 m 90 gm 92 m, 93 m S 21o (g) 1 dc (m) Bio (111) 94 g, 112 gm 531 m, 122g gm 94 Yeast extract-malt extract (ISP-2) 2 (g) 2 (m) 2 (m) gm gm P 1 co (m) 2 go (g) 2 go (g) 121 m, 122 g 90 gm 90 gm 1. 32 6? a e :2 2. 1:12.112 Oatmeal ISP-3 e g 2 c g go 3 gm {P 2 do (2) 1 do (111) 1 ca (2) 93 gm 121 m, 122 g 104 g, 121 g h 3 l; 3? E g 2 5 ea gm 94 51.? gm 33 .5%? Inor anic-salts stare ISP-4 2 c m e 3 go m m g {P 1 eh (m) 2 dc (g) 1% es (111) 121 gm gm 90 gm, 93 m S 219 (m) b (m) 2 eg (In) 94 g .112 gm 263 m, 264 g 92 m, 93 gm Glycerol-asparagine (ISP-5) {1 2 go (g) 2 eh (m) 1 ca (g) in 92 m, 93 gm 104 2, 121 m S=Surface; R=Reve1ae; P=Pi ent' m) =matte; (g)=glossy. 1 Jacobson, E., W. C. Granviilg dnd. E. Foss. 1948. Color harmony manual, 3rd ed. Container Corporation of America,

Chicago, Illinois.

Kelly, K. L., and D. B. Judd. 1955. The ISCC-NBS method of designating colors and a dictionary of color names. US. Dept.

omm. Circ. 553.

See next page for color names.

Color Code for Table 2 yzed yzed yzed sabou- S white white white Color Harmony Manual, roud's R yellow-tan- 3rd ed. 1948 NBS Circular 553, 1955 orange yellowmmomnge ycuow Color Color Name Color Color Namedc p none none none p Chip trose 5 Benn- S Heavy gray-white l lt tyygray-white fair gray-white ett's R yellow yellow yellow b oyster while 263m :vhgte p none none [an 8 t gray Cza S heav ra heav ra trace ra C 8 Sl y 8 "8 B y elts R gray y a y gray y g y gray 8 y lca pale yellow 104g pale greenish yellow sub p none none none 121m pale yellow green rose lcb parchment l2lgm pale yellow green S waymhite graywhim f i gray ldc putty, griege 121m pale yellow green 08* R yeuow yellow cream 122g grayish yellow green P not": on: "one lec light citron gray, 121m pale yellow green one putty- 122g grayish yellow green pew lfe griege, citron gray 112m light olive gray 122g g y y l yeast S no aerial growth no aerial growth white lgc dusty yellow 102g moderate greenish yellow 5 I: o compact to compact w v compact to E Breemsh yellow flocculent flocculent flocculent P 8" I y 7 growth at base growth at base growth at base 93m yell wi h a nitrate reduced nitrate not rednitrate not red- Zeb to nitrite uced to nitrite uced to nitrite 933m yellowish gray Nut- S no surface no surface trace white aer- 2 dc naturaLstring 93gm yellowish gray rient graylsh yellow nitrate growth growth ial rowth on Oatmeal, Sand sur ace pellicle 2fe covert gray 94g light ol ve brown P one none none 1 8 8 9 8") O compact to floccompact to floccompact to floc- 8 bambooi chalnms 3 E culent growth at culent growth at culent growth at 2ge covert tan, griege 94in light olive brown base base base grayish I nitrate not rednitrate not rednitrate reduced 2m y?! .q grfiylsh yellow I uced to nitrite uc'ed to nitrite to nitrite 94g llght ollve brown Lit- S trace gray aeritan surface ring gray-white aeri- 8,. ghk lYq mus al growth on al gr wth n 21g slate tan 1 10g gray sholive milk Surface i bl surface 1 12m light olive gray I i 211mm) 0 e tonization e tonization peptonization Jfe silver y 2 light brownish s y gosiplete. gal lial. pH 6.6 complete.

pH 6.5 pH 7.75 TABLE 3 Cultural Characteristics of S. hygroscopicus Cultures S sum Agar S. hygroscopic S. hygrorcopi- S. endu: Medivar. enhygrus cus CBS NRRL 2339 B ium O Other characteristics TABLE 4 Pep- S trace white very slight pale gray white tone S trace white "on R yellow yellow yellow-4a Utilization of Carbon Compounds in. the Synthetic P melanin negative melanin negative melanin negative 40 Mcdlum of Pndham and Gotflleb Cal- 8 trace white trace white trace white ciurri R colorless colorless colorless S. hygroscopicii: S. hygroscapicus S endus mal- P none none none var. enhygrus CBS RRL ate 0 malate not malate not malate not 2339 solubilized solubilized solubilized Control Glucose S gray-white gray-white pale gray white aspa- R cream gray cream yellow-pink l. D-xylose ragine P none none none 2. L-arabinose Skim S gray-pink-white gray-pink-white fair gray-white 3. rhainnose milk R Yellow-pink-tan yellow-pink-tan yellow 4. D-fructose P yellow-pink yellow-pink yellow 5. D-galactose 0 Casein solubi- Casein solubilcasein solubil- 6, D-glucose ilized around ized around ized 7. D-mann0se growth growth H 8. maltose Tyr- S gray gTay gray-white 9. sucrose (l osine R red-tan red-tan yellow l0. lactose P red-tan red-tan pale yellow ll. cellobiose O tyrosine tyrosine tyrosine l2. raffinose solubilized solubilized solubilized l3, d xtri Xan- S gray-white trace gray-white gray-white 5 5 l4. inulin thine R pale yellow pale yellow yellow 15. soluble starch P none none none l6. glycerol 0 xanthine not xanthine not xanthine not 17. d lci l solubilized solubilized solubilized l8. D-mannitol Yeast S 'cream-gray-whitecream-gray-white cream-gray-wliite l9. D-sorbitol ("l extrawith moist black with moist black 20, DJ imI ct malt patches patches 2 l salicin extra- R yellow-olive yellow-olive gray-brown 22. phenol ct P none none none 23. cresol casein S gray gray gray 24. Na formate ("l starch Rp gray gray gray v 25. Na oxalate none none none 0 starch hydrolstarch hydrolstarch hydrol- Na tin-Pram yzed yzed Wed 7. Na salicylate Nut- S white white white Na F rient R cream cream cream Na 7* starch P pale yellow pale yellow pale yellow Na Succma O starch hydrolstarch hydrolstarch hydrolpositive utilization positive utilization slight growth slight growth no utilization no growth *Pridham, T. 6., and D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 56:l07-l 14.

TABLE Utilization of Carbon compounds in the Modified Medium of Pridham and Gottlieb S hygroscopicus var. enhygrus S. endus NRRL Growth similar to or less than growth on basal medium without carbon compound. Vegetative growth equal to or more than with glucose.

Vegative growth significantly better than on basal medium without carbon compound, but somewhat better than with glucose. i Doubtful. Growth only slightly better than on basal medium without carbon compound and significantly less than with glucose. *Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Stggpgmgmaes species. International Journal of Systemic Bacteriology. l

The new compound of the invention is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions.

It is to be understood also that for the preparation of limited amounts surface cultures and bottles can be 4 employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include com steep liquor, yeast, autolyzed brewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distillers solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as media components.

Production of the compound of the invention can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18 and 40 C., and preferably between about 20 and 32 C. Ordinarily, optimum production of the compound is obtained in about 2 to days. The medium normally remains basic during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the new compound and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When ayoung, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the 5 production of the new compound,as long as it is such that a good growth of the microorganism is obtained.

The new compound of the invention is an ampotheric, non-polyenic chemical compound. It is soluble at a concentration of less than 5 mg./ml. in water, acetone, ethyl acetate, methyl ethyl ketone, methylene chloride, chloroform, l-butanol, and ether. It is soluble at a concentration of greater than 10 mg./ml. in methanol,

ethanol, ,l-propanol, glacial acetic acid, acetone-water (3:2) and water-saturated l-butanol. v

A variety of procedures can be employed in the isolation and purification of enhygrofungin, for example,

solvent extraction, partition chromatography, silica gel 0 mycin. The filtration is preferably carried out in the 45 taining filter cake by means of a low molecular weight alcohol. Methanol or ethanol can be used in the anhydrous form but the propyl' and butyl alcohols are poor solvents for endomycin unless they are mixed with water. The preferred extracting solvents are mixtures of the lower molecular weight aliphatic alcohols and water, containing from thirty to ninety percent of the alcohol and ten to seventy percent of water, the optimum mixture being dependent upon the particular alcohol used for extraction.

' Enhygrofungin can be separated from the other components of the endomycin complex by partition chromatography using a solvent system consisting of methyl ethyl ketonezethyl acetate:water (10:15: 1 .5). Fractions from the column can be analyzed by thin-layer chro- 0 matography using 10 X 20 cm. glass plates prepared with silica gel HF, (E. Merck, A.G.-Darmstadt, Ger

of methanol (20-fold concentration). Ten microliters is applied to the plate and developed with the solvent system methyl ethyl ketonezacetonezwater ([50:50:34). After development, the plates are air dried then sprayed with a freshly prepared mixture composed of anisaldehydez95 percent ethanolzconcentrated sulfuric acidzglacial acetic acid 0.5:9.0:0.5:0.l (ml.)] and heated to 90-l C. for -10 minutes. En-

hygrofungin (rf approx. 0.23 appears as a dark blue.

spot.

Enhygrofungin can be recovered from partition column fractions containing only enhygrofungin, as

shown by thin-layer chromatography, by mixing the fractions with Skellysolve B (isomeric hexanes); separating the. phases and mixing the upper phase with water; again separating the phases; adjusting the aqueous phase to about pH 3.8 with, concentrated hydrochloric acid; then mixing with water, filtering and cooling to about5 C..to induce crystallization of enhygrofungin. The resulting crystals can be recrystallized from an acetonezwater (3:2) solution which is adjusted to an acidic pH, as above, filtered and the filtrate stored at/about 2 C. to induce crystallization of enhygrofungin.

An alternate procedure for separation of en- 'hygrofungin from theother components of the endofreeze=dried preparation of enhygrofungin dissolved in acetonezwater (3:1), which is then mixed with water and. refrigerated to induce crystallization of r enhygrofungin. I

Enhygrofungin can be purified by successive transfers from protonated to non-protonated forms and vice versa, especially with other types of treatments intervening as, for: example, solvent extractions and washings, chromatography, and fractional liquid-liquid extraction. In this manner, salts of enhygrofungin can be employed to isolate or upgrade the antibiotic. For example, the antibiotic can be converted to an insoluble salt, such as the picrate, which can be subjected to purification procedures and then used to regenerate the antibiotic. Or the antibiotic can be converted to a water-soluble salt, such as the hydrochloride or sulfate, and the aqueous solution of the salt extracted with variouswater-immiscible solvents before regenerating the antibiotic.

Since enhygrofungin is an ampotheric substance, it

forms l s h- ..a .k i eta lk l ssatth. metals, and amines. Metal salts can be prepared by dissolving enhygrofungin in water, adding a dilute metal base until the pH of the solution is about 7 to 8, and freeze-drying the solution to provide a dried residue consisting of the enhygrofungin metal salt. Enhygrofungin metal salts include the sodium, potassium, and call neutralizingenhygrofungin with the appropriate acid to cium salts. Amine salts of enhygrofungin, including those with organic bases such as primary, secondary, and tertiary mono-, di-, and polyamines also can be formed using the above-described or other commonly employed procedures. Other salts are obtained with therapeutically effective bases which impart additional therapeutic effects thereto. Such bases are, for exam-' ple, the purine bases such as theophyllin, theobromin, caffein, or derivatives of such purine bases; antihistaminic bases which are capable of forming salts with weak acids; pyridine compounds such as nicotinic acid amide, isonicotinic acid hydrazide, and the like; phenylalkylamines such as adrenalin, ephedrin, and the like; choline, and others.

Acid salts of enhygrofungin can be made. by

below aboutpl-l 7.0, and advantageously to about pH 2 to pH 6. Suitable acids for this purpose include hydrochloric, sulfuric, phosphoric, and the like. Acid and base salts of enhygrofungin can be usedfor the same biological purposes as the parent compound.

Enhygrofungin is active against Bacillus subtilis and can be used to minimize or prevent odor in fish and fish crates caused by this organism. It also can be used for treating breeding places of silkworms to prevent or minimize infections caused by B. subtilis. Also, since :enhygrofungin is active against Cryptococcus neoformans, it can be used to treat pigeon roosts to inhibit this fungus which has been found .in pigeon droppings. (J.

Am. Med. Assoc., V0. 191, No. 4, Jan. 25, 1965, pp. 269-274). Also, the novel antibiotic of the invention can be used to swab laboratory benches and equipment in a mycological laboratory.

Hereinafter are described non-limiting examples of ;the process and products of the present invention All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 A. Fermentation A soil stock of Streptomyces hygroscopicus var. en-

hygrus, NRRL 3664, is used to inoculate a series of 500-ml. Erlenmeyer flasks, each containing ml. of sterile seed medium consisting of the following ingredients:

Glucose monohydrate 25 g./l.v Pharmamedia 25 g./l. Tap water Balance Pharmamedia is an industrial grade of cottonseed flour produced by l Trader's Oil Mill Company. Fort Worth, Texas.

Glucose monohydrate 60 g./l. Hi-starch" 20 g./l. Calcium carbonate 4 g./l. Sodium nitrate 4 g./l. Pharmamedia 20 g./l. Tap water-q.s. Balance The pHof the fermentation medium is adjusted to 7.2 with an aqueous solution of sodium hydroxide before sterilization.

Dextrose 10.0 g.ll. Yeast extract 2.5 g./l. -KH,PO, 1.0 g./l. Agar 17.5 g./l.

Petri dishes are poured and a 12.7 mm. paper disc is placed upon the solidified agar. An 0.08 ml. of aliquot of the enhygrofungin preparation is applied, and the.

zone of inhibition of growth is determined after incubation for 18 hrs. at 28 C.

The antibiotic activity of the fermentation beer is expressed in terms of endits. In order to convert endits to micrograms, the following formula is used: 1 mcg. 4 endits. A controlled fermentation yield as assayed by S. cerevisiae, as described above, ranges from 4 to 5 mg. of antibiotic/ml.

It is noted that this assay represents the sum of the active endomycin complex components present. Assays for enhygrofungin are not specific until the enhygrofungin is separated from the other components by partition chromatography, as described above and infra.

B. Recovery,

The whole beer from an enhygrofungin fermentation, as described above, (2,800 ml. assaying 7,700 en? dits/ml. against S. cerevisiae) is adjusted to pH 4.5 with hydrochloric acid. The acidified beer is then filtered using diatomaceousearth as filter aid. The mycelial cake is extracted 3 times with 700-ml. each of l-butanol saturated with water at 50 C. The butanol is diluted with 2 volumes of Skellysolve B. The oily precipitate which forms is collected, dissolved in 80 percent ethanol and evaporated under vacuum to dryness; yield 1.73 g. assaying 2,100 endits/mg. against S. cerevisiae.

C. Purification 1 An impure preparation containing enhygrofungin, obtained as described above, (75 g. assaying 250 mcg./mg. against S. cerevisia e) is subjected to partition chromatography. The partition column is prepared as follows:

Dicalite 4200 (3,000 g., Great Lakes Carbon Corporation) is mixed with approximately 35 liters of upper phase solvent and 1,200 ml. of lower phase solvent. This upper phase solvent and lower phase solvent is prepared by mixing the following solvents and separating the phases: methyl ethyl ketone 1,000 parts, ethyl acetate 500 parts, water 150 parts. The mixture is poured into a cm. diameter (ID) glass column and the Dicalite is packed to a constant height (137 cm) with down flowing upper phase solvent. The solvent level is drained to about 1 inch above the packed column bed.

The enhygrofungin preparation described above, (75

5 g., 250 mcg./mg.) is dissolved in 120 ml. of lower phase solvent. This solution is mixed with 240 g. of Dicalite 4200 and enough upper phase solvent to make the mixture fluid. This mixture is poured ontothe top of the prepared column bed and the solvent level is drained to the level of the newly added Dicalite mixture. Fresh upper phase solvent is added and the column is developed at a flow rate of 16 liters/hour. Four-liter fractions are collected after the introduction of the load. Column fractions and preparations are analyzed by thin-layer chromatography, prepared as described above. Analyses of the column fractions by thin-layer chromatography show that fractions 9 through 23 contain the major share of purified enhygrofungin. These fractions are combined (60 liters) and mixed with 30 liters of Skellysolve B. The phases are'separated and the upper phase is mixed with 500 ml. of water. The

phases are again separated and the washed upper phase is discarded, but the aqueous phases are combined (4,300 ml.), adjusted to pH 3.8 with concentrated hydrochloric acid (0.3 ml.), mixed with water (5% volume, 2,150 ml.), filtered, cooled, and held at a temperature of 5 C. until crystallization is complete. En-

hygrofungin crystals are removed by filtration, washed 5 with water and dried in vacuo to a constant weight; yield, 10.5 g. of white enhygrofungin crystals.

A portion of the enhygrofungin crystals, obtained above, (8.64 g.) are recrystallized by dissolving in 165 ml. of acetone:water (3:2) and the solution is adjusted to pH 4.0 with hydrochloric acid, then filtered. The clear filtrate is mixed with 1,500 ml. of filtered water until crystallization of enhygrofungin crystals begins. The mixture is then stored overnight at 2 C. The enhygrofungin crystals are removed by filtration, washed with water (25 ml.), and dried in vacuo to a constant weight; yield, 8.3 g. of highly pure enhygrofungin crystals. 1

Example 2 Enhygrofungin is recovered from a preparation of the endomycin complex using the following silica gel chromatography procedure. Silica gel (Merck A.G., Darmstadt, 5 kg.) is mixed with Skellysolve B, poured into a 10 cm. (ID) glass chromatography tube, and

packed to a constant height (122 cm.) with flowing LII Skellysolve B. A preparation of endomycin complex (25 g.), prepared as described in Example 1, Parts A and B, is dissolved in ml. of methanol, and mixed with 200 g. of silica gel. The solvent is removed by evaporation with circulating air. The resulting dried mixture is poured into a layer of Skellysolve B remaining on top of the silica gel column bed. The level of Skellysolve B is lowered to the level of the load, and

fresh solvent consisting of methyl ethyl ketonezacetonezwater (l50:50:28) is introduced and used to develop the column at a flow rate of @QRilQlllFPPElLZ? n a S b t t a P hygrofungin. These fractions are combined, concentrated in vacuo to an aqueous pure solution which is freeze-dried; yield, 12.5 g. of substantially pure enhygrofungin. A portion of this preparation (50 mg.) is dissolved in 8 ml. of acetonezwater (3:1), mixed with 5 8 ml. of water, and refrigerated until crystallization is complete. The crystals of enhygrofungin are removed by filtration, washed with water, and dried in vacuo to a constant weight; yield, 39 mg. of enhygrofungin assaying 930 meg/mg. against S.cere t isiqe Example 3 The fermentation broth from an tion, as disclosed in Example 3 of US. Pat. No. 2,746,902, can be used as the starting material to prepare enhygrofungin by the procedure of Example 1, supra.

We claim: I

l. Antibiotic enhygrofungin which is active against various Gram-positive bacteria and fungi, and, which, in its essentially pure crystalline form has a. white crystals I b. an elemental analysis: C, 60.95; H, 9.00; N, 4.01;

c. an optical rotation [01],, +20 (0, 0.5 percent in dimethylformamide);

d. amelting point ofabout ll9.3 C.;

e. solubilities as follows: soluble at a level of less than 5 mg ./ml. in water, acetone, ethyl acetate, methyl ethyl ketone, methylene chloride, chloroform, lbutanol, and ether;' soluble at a concentration greater than 10 mg./ml. in methanol, ethanol, npropanol, glacial acetic acid, acetone-water (3:2), and water saturatedl -butanol;

S. endus fermentaf. a characteristic infrared absorption spectrum as shown in FIG. 1 of the accompanying drawing; and,

g. a characteristic paper chromatographic patternas shown in FIG. 2 of the accompanying drawing, and being substantially free'of other components in the endomycin complex.

2. A compound as defined in claim 1, enhygrofungin,

in its essentially pure form.

3. A compound as defined in claim 1, enhygrofungin, in its essentially pure crystalline form.

4. A compound selected from the group consisting of enhygrofungin, according .to claim 1, and a salt thereof with an alkali metal, an alkaline earth metal, or an amine.

5. Acid addition salt of enhygrofungin, the compound defined in claim 1.

6. A process, which comprises cultivating Streptom yces hygroscopicus -var. enhygrus in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen, under aerobic conditions, until substantial activity against Saccharomyces cerevisiae is imparted to said medium by the production of endomycin complex, and isolating enhygrofungin substantially free of other components of the endomx iz complet- A.

7. A process, according to claim 6, in which the isolation comprises filtering the medium, extracting the endomycin complex with a solvent for endomycin, subjecting said extracted endomycin complex to partition chromatography, and isolating pure enhygrofungin from fractions obtained from said partition chromatography. 

2. A compound as defined in claim 1, enhygrofungin, in its essentially pure form.
 3. A compound as defined in claim 1, enhygrofungin, in its essentially pure crystalline form.
 4. A compound selected from the group consisting of enhygrofungin, according to claim 1, and a salt thereof with an alkali metal, an alkaline earth metal, or an amine.
 5. Acid addition salt of enhygrofungin, the compound defined in claim
 1. 6. A process, which comprises cultivating Streptomyces hygroscopicus var. enhygrus in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen, under aerobic conditions, until substantial activity against Saccharomyces cerevisiae is imparted to said medium by the production of endomycin complex, and isolating enhygrofungin substantially free of other components of the endomycin complex.
 7. A process, according to claim 5, in which the isolation comprises filtering the medium, extracting the endomycin complex with a solvent for endomycin, subjecting said extracted endomycin complex to partition chromatography, and isolating pure enhygrofungin from fractions obtained from said partition chromatography. 